Method and apparatus for maintenance and expansion of hemopoietic stem cells and/or progenitor cells

ABSTRACT

A method of preparing a stromal cell conditioned medium useful in expanding undifferentiated hemopoietic stem cells to increase the number of the hemopoietic stem cells is provided. The method comprising: (a) establishing a stromal cell culture in a stationary phase plug-flow bioreactor under continuous flow on a substrate in the form of a sheet, the substrate including a non-woven fibrous matrix forming a physiologically acceptable three-dimensional network of fibers, thereby expanding undifferentiated hemopoietic stem cells; and (b) when a desired stromal cell density has been achieved, collecting medium from the stationary phase plug-flow bioreactor, thereby obtaining the stromal cell conditioned medium useful in expanding the undifferentiated hemopoietic stem cells.

This Application is a continuation-in-part of U.S. patent applicationSer. No. 09/890,401, filed on Jul. 31, 2001, which is a National Phaseof PCT Patent Application No. PCT/US00/02688, filed on Feb. 4, 2000,which claims the benefit of priority of U.S. Provisional Application No.60/118,789, filed on Feb. 4, 1999, the contents of which are allincorporated by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to a method and apparatus for maintenanceand expansion of hemopoietic stem cells. More particularly, the presentinvention relates to a three dimensional stromal cell plug flowbioreactor for the maintenance and/or expansion of hemopoietic stemcells and/or for the production of a conditioned medium for themaintenance and/or expansion of hemopoietic stem cells.

The hemopoietic system in mammals is composed of a heterogenouspopulation of cells that range in function from mature cells withlimited proliferative potential to pluripotent stem cells with extensiveproliferative, differentiative and self renewal capacities (1-3).Hemopoietic stem cells (HSC) are exclusively required for hemopoieticreconstitution following transplantation and serve as a primary targetfor gene therapy. In spite of the key role of stem cells in maintainingthe hemopoietic system, their extremely low frequency in hemopoietictissue, as well as the limited ability to maintain or expandundifferentiated stem cells under ex-vivo conditions for prolongedperiods of time, not only remains a major drawback to essential clinicalapplications of these cells, but also reflects the currentunavailability of, and the need for, novel stem cell regulators.

It is widely accepted that stem cells are intimately associated in vivowith discrete niches within the marrow (4-6), which provide molecularsignals that collectively mediate their differentiation and selfrenewal, via cell-cell contacts or short-range interactions (7). Theseniches are part of the “hemopoietic inductive microenvironment” (HIM),composed of marrow stromal cells, e.g., macrophages, fibroblasts,adipocytes and endothelial cells (8). Marrow stromal cells maintain thefunctional integrity of the HIM by providing extracellular matrix (ECM)proteins and basement membrane components that facilitate cell-cellcontact (9-11). They also provide various soluble or resident cytokinesneeded for controlled hemopoietic cell differentiation and proliferation(12-14).

In view of the above, it is not surprising that efforts to developculture systems for the prolonged maintenance of human HSC were mainlyfocused on the use of pre-established primary marrow stromal cellmonolayers. These included long-term cultures of unirradiated (Dextercultures, 15) or irradiated (16-19) primary human marrow stroma, as wellas human or murine stromal cell lines (16, 19-24), with or withoutexogenously added cytokines. Output assays for HSC initially relied onthe capacity of such cells to produce myeloid progeny (long-term cultureinitiating cells; LTC-IC) or to generate colonies with cobblestonemorphology (cobblestone area forming cells; CAFC) after prolongedculture (5-7 weeks) on such stromal cells (16,17). In spite of thewidespread use of LTC-IC and CAFC assays, it is becoming increasinglyobvious, however, that they detect highly primitive progenitors, ratherthan true repopulating hemopoietic stem cells (25, 26).

A recently developed human stem cell assay detects a SCID repopulatingcell (SRC), which homes to the bone marrow of non-obese diabetic(NOD)/SCID mice (27), where it gives rise to human myeloid, lymphoid,erythroid and CD34+ progenitor populations (28-30). The SRC isexclusively found in hemopoietic cell fractions expressing the CD34+38−surface antigen (31) and its frequency in CB (1/3×10⁵ cells) is enrichedas compared to BM (1/9×10⁵ cells) or mobilized PB (1/6×10⁶ cells) (32).Very recent studies showed that the SRC resides within a subpopulationof CD34+/38−/CXCR4+ cells (33). CXCR4, a surface receptor for thechemokine stromal cell-derived factor 1 (SDF-1, 34), is apparentlyessential for homing and engraftment of human hemopoietic stem cells inthe NOD/SCID marrow (33). Studies aimed at inducing prolongedmaintenance/expansion of human HSC on stromal cell cultures were mainlybased on CAFC, LTC-IC or the CD34+38− phenotype as end-point assays (16,19-24). Rare reports of SRC maintenance/expansion in stromal cellcultures fail to indicate significant long-term support. For example,allogeneic human marrow stroma was found to induce short-term (7-day)SRC maintenance, followed by a rapid, marked decline (6-fold) inactivity (26). The inability to support the long-termmaintenance/expansion of transplantable human stem cells on stromal celllayers, may be attributed to several factors related to in vitrocultures of these cells. Among these, one may include the use of stromalcell monolayers, which do not reflect the in vivo growth conditionswithin the natural, three-dimensional structure of the bone marrow. Suchconditions may diminish the capacity of stromal cells to provide theoptimal, appropriate supportive microenvironment, as well as thecapacity of stem cells to localize in specific niches and to physicallyinteract with stromal cells and their products. Indeed, evidence for theimportance of a three dimensional (3D) structure for the biologicalactivity of hemopoietic progenitor cells, is provided by the superiorgrowth of a human hemopoietic cell line on stromal cells seeded in a 3Dcollagen matrix, as compared to their proliferation on monolayers ofsuch cells (35). More importantly, a 3D tantalum-coated porousbiomaterial, was recently shown to enhance the short-term maintenance ofmacaque LTCIC or CD34+38− cells, as compared to cells cultured alone oron marrow stromal cell monolayers (36). The effect of stromalcell-coated 3D carriers, was, however, not investigated.

Recent studies have shown the murine AFT024 cell line to be superiorthan human stroma, in supporting 2-3 week survival and maintenance(albeit not expansion) of human CB SRC (37). This line has been found toexpress several novel HIM genes encoding membrane-bound proteins (21,38, 39), which may have an essential role in stem cell physiology. Thepossible expression of these and other genes by stromal cells underconditions which more closely mimic their 3D marrow microenvironment,and thus enable their optimal, physiological functional activity, hasyet to be determined.

Extensive studies have shown that stroma non-contact cultures (19, 21,22, 40, 41) or stroma conditioned media (SCM) (21, 4244), alone or withcytokines, can support the ex-vivo maintenance or expansion of primitivehemopoietic progenitors. SCM has also been shown to improve the recoveryand transduction efficiency of such cells (45, 46). While these findingsagain stress the importance of soluble stromal cell factors, the use ofLTC-IC, CAFC or CD34+38− end-points in such assays cannot reflect theeffect of SCM on maintenance/expansion of transplantable HSC.Furthermore, it is not known whether such SCM, obtained from monolayercultures of stromal cells, indeed contains all stromal cell-associatedgene products involved in human HSC physiology.

Recent attention aimed at ex-vivo expansion of transplantablehemopoietic stem cells has focused on the establishment ofcytokine-supplemented suspension cultures (47-53). These studies havehelped identify the major relevant cytokines for this process, e.g.,early-acting ones such as stem cell factor (SCF), FLT3 ligand andthrombopoietin (TPO). Nevertheless, variable results have been obtained,indicating short-term loss (48, 49), maintenance (50-52) but also somerare examples of SRC expansion, following during 2-4 weeks of culture(47, 53). The interactive capacity of these cytokines and stromal cells,under 3D growth conditions, to support the maintenance/expansion of SRC,has not yet been defined.

There is thus a widely recognized need for, and it would be highlyadvantageous to have, a method and apparatus for ex-vivo expansionand/or maintenance of transplantable hemopoietic stem cells devoid ofthe above limitations, with superior results as is compared to theteachings of the prior art.

SUMMARY OF THE INVENTION

While reducing the present invention to practice, a plug flow bioreactorsystem which closely mimics the 3D bone marrow microenvironment andwhich is capable of supporting the growth and prolonged maintenance ofstromal cells, has been developed. The latter were seeded on porrosiveinorganic carriers made of a non woven fabric matrix of polyester (54),enabling the propagation of large cell numbers in a relatively smallvolume. The structure and packing of the carrier have a major impact onoxygen and nutrient transfer, as well as on local concentrations andreleased stromal cell products (e.g., ECM proteins, cytokines, 55). Inaddition, the capacity of stromal cells cultured in this system topromote the maintenance/expansion of transplantable human hemopoieticstem cells via direct cell-cell contact has been determined to be farsuperior over prior art methods. Furthermore, the capacity ofconditioned medium of stromal cells cultured in this system to promotethe maintenance/expansion of transplantable human hemopoietic stem cellsvia novel stromal-cell associated stem cell factors included therein,has been determined to be far superior over prior art methods.

Thus, according to one aspect of the present invention there is provideda method of expanding undifferentiated hemopoietic stem cells orprogenitor cells, the method comprising the steps of (a) obtainingundifferentiated hemopoietic stem cells or progenitor cells; and (b)seeding the undifferentiated hemopoietic stem cells or progenitor cellsinto a stationary phase plug-flow bioreactor in which a threedimensional stromal cell culture has been preestablished on a substratein the form of a sheet, the substrate including a non-woven fibrousmatrix forming a physiologically acceptable three-dimensional network offibers, thereby expanding undifferentiated hemopoietic stem cells orprogenitor cells.

According to still further features in the described preferredembodiments the method further comprising the step of isolating theundifferentiated hemopoietic stem cells or progenitor cells.

According to another aspect of the present invention there is provided amethod of expanding undifferentiated hemopoietic stem cells orprogenitor cells, the method comprising the steps of (a) obtainingundifferentiated hemopoietic stem cells or progenitor cells; and (b)culturing the undifferentiated hemopoietic stem cells or progenitorcells in a medium containing a stromal cell conditioned medium, thestromal cell conditioned medium being derived from a stationary phaseplug-flow bioreactor in which a three dimensional stromal cell culturehas been established on a substrate in the form of a sheet, thesubstrate including a non-woven fibrous matrix forming a physiologicallyacceptable three-dimensional network of fibers, thereby expandingundifferentiated hemopoietic stem cells or progenitor cells.

According to yet another aspect of the present invention there isprovided a method of preparing a stromal cell conditioned medium usefulin expanding undifferentiated hemopoietic stem cells or progenitorcells, the method comprising the steps of (a) establishing a stromalcell culture in a stationary phase plug-flow bioreactor on a substratein the form of a sheet, the substrate including a non-woven fibrousmatrix forming a physiologically acceptable three-dimensional network offibers, and (b) when a desired stromal cell density has been achieved,collecting medium from the stationary phase plug-flow bioreactor,thereby obtaining the stromal cell conditioned medium useful inexpanding undifferentiated hemopoietic stem cells or progenitor cells.

According to still another aspect of the present invention there isprovided a method of transplanting undifferentiated hemopoietic stemcells or progenitor cells into a recipient, the method comprising thesteps of (a) expanding the undifferentiated hemopoietic stem cells orprogenitor cells by (i) obtaining undifferentiated hemopoietic stemcells or progenitor cells; and (ii) seeding the undifferentiatedhemopoietic stem cells or progenitor cells into a stationary phaseplug-flow bioreactor in which a three dimensional stromal cell culturehas been pre-established on a substrate in the form of a sheet, thesubstrate including a non-woven fibrous matrix forming a physiologicallyacceptable three-dimensional network of fibers, thereby expandingundifferentiated hemopoietic stem cells or progenitor cells; and (b)transplanting the undifferentiated hemopoietic stem cells or progenitorcells resulting from step (a) in the recipient.

According to still further features in the described preferredembodiments the method further comprising the step of isolating theundifferentiated hemopoietic stem cells or progenitor cells prior tostep (b).

According to an additional aspect of the present invention there isprovided a method of transplanting undifferentiated hemopoietic stemcells or progenitor cells into a recipient, the method comprising thesteps of (a) expanding the undifferentiated hemopoietic stem cells orprogenitor cells by (i) obtaining undifferentiated hemopoietic stemcells or progenitor cells; and (ii) culturing the undifferentiatedhemopoietic stem cells or progenitor cells in a medium containing astromal cell conditioned medium, the stromal cell conditioned mediumbeing derived from a stationary phase plug-flow bioreactor in which athree dimensional stromal cell culture has been established on asubstrate in the form of a sheet, the substrate including a non-wovenfibrous matrix forming a physiologically acceptable three-dimensionalnetwork of fibers, thereby expanding undifferentiated hemopoietic stemcells or progenitor cells.

According to yet an additional aspect of the present invention there isprovided a bioreactor plug comprising a container having an outlet andan inlet and containing therein a substrate in the form of a sheet, thesubstrate including a non-woven fibrous matrix forming a physiologicallyacceptable three-dimensional network of fibers, the substrate supportingat least 5×10⁶ stromal cells per cubic centimeter of the substrate.

According to still an additional aspect of the present invention thereis provided a plug-flow bioreactor comprising the above bioreactor plug.

According to a further aspect of the present invention there is provideda method of expanding undifferentiated hemopoietic stem cells toincrease the number of the hemopoietic stem cells, the method comprisingthe steps of: (a) obtaining the undifferentiated hemopoietic stem cells;and (b) culturing the undifferentiated hemopoietic stem cells in amedium containing a stromal cell conditioned medium, the stromal cellconditioned medium being derived from a stationary phase plug-flowbioreactor in which a three dimensional stromal cell culture has beenestablished under continuous flow on a substrate in the form of a sheet,the substrate including a non-woven fibrous matrix forming aphysiologically acceptable three-dimensional network of fibers, therebyexpanding the undifferentiated hemopoietic stem cells.

According to yet a further aspect of the present invention there isprovided a method of transplanting undifferentiated hemopoietic stemcells into a recipient, the method comprising the steps of: (a)expanding/maintaining the undifferentiated hemopoietic stem cells by:(i) obtaining the undifferentiated hemopoietic stem cells; and (ii)culturing the undifferentiated hemopoietic stem cells in a mediumcontaining a stromal cell conditioned medium, the stromal cellconditioned medium being derived from a stationary phase plug-flowbioreactor in which a three dimensional stromal cell culture has beenestablished under continuous flow on a substrate in the form of a sheet,the substrate including a non-woven fibrous matrix forming aphysiologically acceptable three-dimensional network of fibers, therebyexpanding the undifferentiated hemopoietic stem cells.

According to still a further aspect of the present invention there isprovided a method of preparing a stromal cell conditioned medium usefulin expanding undifferentiated hemopoietic stem cells to increase thenumber of the hemopoietic stem cells, the method comprising: (a)establishing a stromal cell culture in a stationary phase plug-flowbioreactor under continuous flow on a substrate in the form of a sheet,the substrate including a non-woven fibrous matrix forming aphysiologically acceptable three-dimensional network of fibers, therebyexpanding undifferentiated hemopoietic stem cells; and (b) when adesired stromal cell density has been achieved, collecting medium fromthe stationary phase plug-flow bioreactor, thereby obtaining the stromalcell conditioned medium useful in expanding the undifferentiatedhemopoietic stem cells.

According to further features in preferred embodiments of the inventiondescribed below, the undifferentiated hemopoietic stem cells orprogenitor cells are cells isolated from a tissue selected from thegroup consisting of cord blood, mobilized peripheral blood andbone-marrow.

According to still further features in the described preferredembodiments the undifferentiated hemopoietic stem cells or progenitorcells and stromal cells of-the stromal cell culture share common HLAantigens.

According to still further features in the described preferredembodiments the undifferentiated hemopoietic stem cells or progenitorcells and stromal cells of the stromal cell culture are from a singleindividual.

According to still further features in the described preferredembodiments the undifferentiated hemopoietic stem cells or progenitorcells and stromal cells of the stromal cell culture are from differentindividuals.

According to still further features in the described preferredembodiments the undifferentiated hemopoietic stem cells or progenitorcells and stromal cells of the stromal cell culture are from the samespecies.

According to still further features in the described preferredembodiments the undifferentiated hemopoietic stem cells or progenitorcells and stromal cells of the stromal cell culture are from differentspecies.

According to still further features in the described preferredembodiments stromal cells of the stromal cell culture are grown to adensity of at least 5×10⁶ cells per a cubic centimeter of the substrate.

According to still further features in the described preferredembodiments stromal cells of the stromal cell culture are grown to adensity of at least 10⁷ cells per a cubic centimeter of the substrate.

According to still further features in the described preferredembodiments the step of seeding the undifferentiated hemopoietic stemcells or progenitor cells into the stationary phase plug-flow bioreactoris effected while flow in the bioreactor is shut off for at least 10hours following the seeding.

According to still further features in the described preferredembodiments the fibers form a pore volume as a percentage of totalvolume of from 40 to 95% and a pore size of from 10 microns to 100microns.

According to still further features in the described preferredembodiments the matrix is made of fiber selected from the groupconsisting of flat, non-round, and hollow fibers and mixtures thereof,the fibers being of from 0.5 microns to 50 microns in diameter or width.

According to still further features in the described preferredembodiments the matrix is composed of ribbon formed fibers having awidth of from 2 microns.

According to still further features in the described preferredembodiments the ratio of width to thickness of the fibers is at least2:1.

According to still further features in the described preferredembodiments the matrix having a pore volume as a percentage of totalvolume of from 60 to 95%.

According to still further features in the described preferredembodiments the matrix has a height of 50-1000 μm.

According to still further features in the described preferredembodiments the material of the matrix is selected from the groupconsisting of polyesters, polyalkylenes, polyfluorochloroethylenes,polyvinyl chloride, polystyrene, polysulfones, cellulose acetate, glassfibers, and inert metal fibers.

According to still further features in the described preferredembodiments the matrix is in a shape selected from the group consistingof squares, rings, discs, and cruciforms.

According to still further features in the described preferredembodiments the matrix is coated with poly-D-lysine.

According to still further features in the described preferredembodiments the stromal cells comprise stromal cells of primary culture.

According to still further features in the described preferredembodiments the stromal cells comprise stromal cells of a cell line.

According to still further features in the described preferredembodiments the stromal cell conditioned medium is devoid of addedcytokines.

According to still further features in the described preferredembodiments a rate of the continuous flow is in a range of 0.1 to 25ml/minute.

According to still further features in the described preferredembodiments a rate of the continuous flow is in a range of 1 to 10ml/minute.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing more effective means forexpanding/maintaining undifferentiated hemopoietic stem cells.

Implementation of the method and bioreactor of the present invention mayinvolve performing or completing selected tasks or steps manually,automatically, or a combination thereof. Moreover, according to actualinstrumentation and equipment of preferred embodiments of the method andbioreactor of the present invention, several selected steps could beimplemented by hardware or by software on any operating system of anyfirmware or a combination thereof. For example, as hardware, selectedsteps of the invention could be implemented as a chip or a circuit. Assoftware, selected steps of the invention could be implemented as aplurality of software instructions being executed by a computer usingany suitable operating system. In any case, selected steps of the methodand bioreactor of the invention could be described as being performed bya data processor, such as a computing platform for executing a pluralityof instructions.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIG. 1 is a schematic depiction of an exemplary plug-flow bioreactor 20which served while reducing the present invention to practice; 1—mediumreservoir; 2—gas mixture container; 3—gas filters; 4—injection points;5—plug or container of plug flow bioreactor 20; 6—flow monitors; 6a—flow valves; 7—conditioned medium collecting/separating container;8—container for medium exchange; 9—peristaltic pump; 10—sampling point;11—container for medium exchange; 12—0₂ monitor; 14—steering device;PH—pH probe.

FIG. 2 demonstrates CAFC maintenance by 14F1.1 cells. Cord blood CD34+cells were seeded at limiting-dilution onto irradiated 14F1.1 or primaryhuman marrow stroma. Cobblestone formation was determined 5 weeks later.Results represent the mean±SD of 2 independent experiments.

FIG. 3 demonstrates LTC-IC maintenance by 14F11. cells. Cord blood CD34+cells were seeded at limiting dilution onto irradiated 14F1.1 or primaryhuman marrow stroma. Myeloid colony formation was determined 7 weekslater. FLT-3 ligand (300 ng/ml), TPO (300 ng/ml) and SCF (100 ng/ml)were added with weekly medium replacement. Results represent the mean±SDof 2 experiments.

FIG. 4 demonstrates expansion of CD34+38− cells on 14F1.1 and primaryhuman marrow stroma. CD34+ cells were seeded onto 14F1.1 or human marrowstroma at 70 CD34+38− cells/well. Cytokines were added weekly. Cultureswere trypsinized 7-21 days later. CD34+38− were determined by FACSanalysis. Results represent the mean±SD of 2 independent experiments.

FIGS. 5 a-b show scanning electron micrographs (SEM) of carrier seededwith 14F1.1 stromal cell line following 10 days (FIG. 5 a) or following40 days (Figure b). Magnification: ×150.

FIGS. 6 a-b demonstrate the effect of 3D versus 2D 14F1.1 conditionedmedium on CD34+38− expansion. CD34+ cells were seeded in suspensioncultures in the presence of various concentrations of conditioned mediumfrom 14F1.1 and primary human marrow stroma. CD34+38− cell numbers weredetermined by FACS analysis. Results represent the mean±SD of 2independent experiments.

FIG. 7 demonstrates maintenance of CD34+38− cells on stromal-cell coatedcarriers. Stromal cell-coated carriers were removed from the 3D systeminto silicone-coated 96-well dishes, followed by addition of 1.5×10⁴CD34+ cells. Controls contained carriers alone and carrier-equivalentnumbers of monolayer (2D) grown 14F1.1 cells. Cells were harvested atthe designated times and analyzed by FACS. Results represent the mean±SDof 2 independent experiments.

FIGS. 8 a-c are bar graphs depicting the ability of primary stromalcells conditioned medium (SCM) obtained from human primary BM cultures,grown on 2D or on 3D supports, to support expansion of hematopoieticprogenitors. FIG. 8 a—shows CD34⁺ cell expansion in the presence ofdifferent culture media. FIG. 8 b—shows CD34⁺ cell expansion in thepresence of different culture media. FIG. 8 c—shows CD34⁺/38⁻CXCR4⁺ cellexpansion in the presence of different culture media.

FIGS. 9 a-c are bar graphs depicting the ability of stromal cellsconditioned medium from murine AFT024 cell cultures, grown on 2D or on3D supports to support hematopoietic progenitor cell expansion. FIG. 9a—shows CD34⁺ cell expansion in the presence of different culture media.FIG. 9 b—shows CD3⁴ ⁺/38⁻ cell expansion in the presence of differentculture media. FIG. 9 c—shows CD34⁺/38⁻CXCR4⁺ cell expansion in thepresence of different culture media.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of methods and bioreactor for hemopoietic stemcell expansion/maintenance which can be used for transplantation in arecipient or for other purposes as if further detailed hereinunder.Specifically, the present invention is of a three dimensional stromalcell plug flow bioreactor for the maintenance and/or expansion ofhemopoietic stem cells and/or for the production of a conditioned mediumfor the maintenance and/or expansion of hemopoietic stem cells, whichcan be used in a variety of applications.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details of construction and the arrangement of the components setforth in the following description or illustrated in the drawings. Theinvention is capable of other embodiments or of being practiced orcarried out in various ways. Also, it is to be understood that thephraseology and terminology employed herein is for the purpose ofdescription and should not be regarded as limiting.

Current strategies aimed at long-term ex-vivo maintenance or expansionof transplantable human hemopoietic stem cells (HSC), have so far beenmet with limited success. A novel three dimensional (3D) plug flowbioreactor which closely mimics the bone marrow microenvironment andwhich is capable of supporting the growth and prolonged maintenance ofmarrow stromal cells is described herein. The latter are seeded onporrosive carriers made of a non woven fabric matrix of polyester,packed in a glass column, thereby enabling the propagation of large cellnumbers in a relatively small volume. In the examples provided in theExample section that follows, the bioreactor was seeded with the murine14F1.1 stromal cell line or alternatively with primary human marrowstromal cells. By day 40 following seeding, the carriers contained a100-fold increased cell density. The density at various levels of thecolumn was the same, indicating a homogenous transfer of oxygen andnutrients to the cells. Media conditioned by stromal cells within thebioreactor (3D SCM) was superior to stromal cell monolayer (2D) SCM, insupporting the long-term maintenance of human cord blood (CB) CD34+38−cells. 3D SCM was also capable of supporting the expansion ofCD34+38−CXCR4+ cells, which represent SCID/NOD repopulating cells (SRC).In the presence of cytokines (FLT3 ligand and TPO), 3D SCM enhanced stemcell self renewal and inhibited differentiation, while the oppositeeffect was induced by 2D SCM+ cytokines. Three dimensional stromal-stemcell cocultures also exhibited superior maintenance of CD34+38− cellsthan cocultures on monolayer stromal cells. These findings demonstratethat the 3D plug flow bioreactor provides a suitable system for ex-vivomaintenance/expansion of human HSC via superior stromal-stem cellcontact and perhaps via stromal cell production of known and/or novelstem cell regulators.

The human HSC is an essential target for transplantation and genetherapy. The highly reduced frequency of HSCs, as well as the currentunavailablity of growth factors capable of inducing stem cell selfrenewal in the absence of terminal differentiation, still provide amajor hindrance to the implementation of such strategies, well as to thelarge-scale setup of HSC “banks”.

Current strategies aimed at long-term maintenance/expansion ofundifferentiated human HSC, have so far been met with limited success.While recent studies with cytokine-supplemented suspension cultures haveshown some SRC expansion, this process was also accompanied by a massiveincrement of early hemopoietic progenitors (53, 62), indicating that asubstantial degree of stem cell differentiation was taking place. Anideal system would be one, for example, in which SRC were expanded,while LTC-IC remain reduced in numbers.

Current systems for hemopoietic cell expansion employ perfusedsuspension cultures of hemopoietic cells, alone (see, U.S. Pat. No.5,646,043) or seeded on stromal-cell monolayers (see, U.S. Pat. No.5,605,822). While the former system demonstrates a tremendous productionof committed progenitors, the latter suffers from the non-physiologicalnature of monolayer stromal-stem cell interactions. Additional systemsfor stem cell expansion describe the use of stromal cell conditionedmedia (U.S. Pat. Nos. 4,536,151 and 5,437,994). However, the latter wereobtained from stromal cell monolayer cultures, which are clearly shownherein to be inferior and different in stem cell activating capacity, ascompared to 3D SCM (see, Table 3 of the Examples section). Although astationary phase bioreactor using stromal cell-coated glass beads hasrecently been described (U.S. Pat. No. 5,906,940), the beads do notprovide a physiological, 3D structure and allow the propagation of a10-fold lower number of stromal cells per ml, as compared to thecarriers employed while reducing the present invention to practice. Theadvantage of a 3D versus monolayer stromal cell culture is clearlydemonstrated by the findings presented herein of the superior capacityof 3D derived SCM or 3D stromal cell cultures to support the maintenanceof CD34+38− cells (see, FIGS. 6 and 7). The superior effect of 3D SCMmay be attributed to enhanced levels of known cytokines or novel stemcell regulators.

Experiments aimed at evaluating the combined effects of 3D SCM andvarious cytokines (SCF, FLT3 ligand, TPO), on CD34+38−CXCR4+ (or SRC)maintenance/expansion (Table 3), clearly show a beneficial effect of 3DSCM. These findings can be attributed to a relative inhibitory effect of3D SCM on stem cell differentiation. These findings strongly indicatethat under 3D conditions, novel stromal cell associated factors which,perhaps less active themselves, may act synergistically with suchcytokines, were produced. The use of LTC-IC and committed progenitorcell (GM-CFU) output readouts, in addition to CD34+ output, allow totest for stem cell differentiation.

The bioreactor described herein is unique in that it combines both 3Dstromal cell cultures with a continuous flow system. While 3Dstromal-hemopoietic cell systems without continuous medium flow haverecently been described (U.S. Pat. No. 5,541,107), the findingsdescribed herein (see, for example, FIG. 7) clearly demonstrate thediminished advantage of 3D stromal cell cultures relative to monolayers,in the absence of continuous flow.

The 3D plug-flow bioreactor described herein is capable of supportingthe long-term growth of stromal cell lines, as well as primary marrowstromal cells. The use of stromal cells in the bioreactor is not onlyessential for the establishment of superior stromal-stem cell contact(via unique “niches” and cell-cell, cell-ECM interactions), but also forstromal cell production of known and novel soluble and membrane-boundcytokines. Stromal cells can facilitate the supplementation of suchbioreactors with appropriate cytokines, by using genetically engineeredcytokine-producing variants.

Bioreactor stromal cells can also be engineered to serve as retroviralpackaging cell lines, enabling the efficient transduction of geneticmaterial into stem cells, within the bioreactor itself. The use ofvarious stromal cells in the bioreactor can also allow the selection ofthe most suitable substrate for purging of Ph-positive stem cells, thelatter known for their lesser capacity for stromal cell adherence (63).Primary stromal cells have the advantage that they enable theestablishment of “autologous” stromal-stem cell bioreactors, on whichautologous or even cord blood stem cells can be expanded and whicheliminate the need to remove stromal cells prior to transplantation.

While the initial seeding experiments in the bioreactor indicated arather small yield of CD34+38− cells in the carrier, the medium flowrate following seeding, as well as initial CD34+ cell numbers seededinto the bioreactor can be readily optimized. CD34+38−CXCR4+ analysis atearly time points (1-4 days) following seeding is essential for suchoptimization.

In sharp distinction to prior art methods, the bioreactor of the presentinvention employs a growth matrix that substantially increases theavailable attachment surface for the adherence of the stromal cells soas to mimic the mechanical infrastructure of bone marrow. For example,for a growth matrix of 0.5 mm in height, the increase is by a factor ofat least from 5 to 30 times, calculated by projection onto a base of thegrowth matrix. Such an increase by a factor of about 5 to 30 times, isper unit layer, and if a plurality of such layers, either stacked orseparated by spacers or the like, is used, the factor of 5 to 30 timesapplies per each such structure. When the matrix is used in sheet form,preferably non-woven fiber sheets, or sheets of open-pore foamedpolymers, the preferred thickness of the sheet is about 50 to 1000 μm ormore, there being provided adequate porosity for cell entrance, entranceof nutrients and for removal of waste products from the sheet. Accordingto a preferred embodiment the pores having an effective diameter of 10μm to 100 μm. Such sheets can be prepared from fibers of variousthicknesses, the preferred fiber thickness or fiber diameter range beingfrom about 0.5 μm to 20 μm still more preferred fibers are in the rangeof 10 μm to 15 μm in diameter.

The structures of the invention may be supported by, or even betterbonded to, a porous support sheet or screen providing for dimensionalstability and physical strength.

Such matrix sheets may also be cut, punched, or shredded to provideparticles with projected area of the order of about 0.2 mm² to about 10mm², with the same order of thickness (about 5 to 1000 μm).

Further details relating to the fabrication, use and/or advantages ofthe growth matrix which was used to reduce the present invention topractice are described in U.S. Pat. No. 5,168,085, and in particular,U.S. Pat. No. 5,266,476, both are incorporated herein by reference.

As will readily be appreciated by the skilled artisan, the presentinvention provides expanded undifferentiated hemopoietic stem cellpopulation which can be used in a variety of applications, such as, butnot limited to: (i) expansion of human stem cells (of autologous or cordblood source) on recipient stroma, without the need for stromal-stemcell separation prior to transplantation; (ii) depletion of Ph+ CML stemcells in an autologous setting via stromal-stem cell interactions; (iii)gene transfer into self-renewing stem cells within the bioreactor orfollowing harvesting from the bioreactor; (iv) production of 3D stromalcell conditioned medium (SCM) for ex-vivo maintenance/expansion ofundifferentiated hemopoietic stem cells in suspension cultures or in astem cell bioreactor; (v) isolation of novel proteins inducing stem cellself renewal in the absence of differentiation, as well as proteinshaving additional biological functions; (vi) isolation of 3D stromalcell RNA for cloning of novel stromal cell-associated stem cellregulators and additional functional stromal cell gene products.

According to one aspect of the present invention there is provided amethod of expanding/maintaining undifferentiated hemopoietic stem cellsor progenitor cells. The method according to this aspect of the presentinvention is effected by implementing the following method steps. First,undifferentiated hempoietic stem cells or progenitor cells are obtained.Second, the undifferentiated hemopoietic stem cells or progenitor cellsare seeded into a stationary phase plug-flow bioreactor, an example ofwhich is depicted in FIG. 1 along with reference numerals, in which athree dimensional stromal cell culture, of either stromal cell line orprimary stromal cell culture, have been pre-established on a substratein the form of a sheet, the substrate including a non-woven fibrousmatrix forming a physiologically acceptable three-dimensional network offibers, thereby, as is further described above and exemplified in theExamples section that follows, expanding/maintaining undifferentiatedhemopoietic stem cells or progenitor cells.

As used herein in the specification and in the claims section thatfollows, the phrase “undifferentiated hemopoietic stem cells” refers touncommitted hemopoietic cells.

As used herein in the specification and in the claims section thatfollows, the phrase “progenitor cells” refers to committed, yet immaturehemopoietic cells.

Both undifferentiated hemopoietic stem cells and progenitor cells areCD34+ cells. Thus, the phrase “obtaining undifferentiated hemopoieticstem cells or progenitor cells” and its equivalent phrase“undifferentiated hemopoietic stem cells or progenitor cells areobtained” both refer to the obtainment of either isolatedundifferentiated hemopoietic stem cells and/or progenitor cells, or apopulation of CD34+ cells which contain undifferentiated hemopoieticstem cells and progenitor cells.

As used herein in the specification and in the claims section thatfollows, the terms “expanding” and “expansion” refer to substantiallydifferentiationless cell growth, i.e., increase of a cell populationwithout differentiation accompanying such increase.

As used herein in the specification and in the claims section thatfollows, the terms “maintaining” and “maintenance” refer tosubstantially differentiationless cell renewal, i.e., substantiallystationary cell population without differentiation accompanying suchstationarity.

As used herein the term “differentiation” refers to change fromrelatively generalized to specialized kinds during development. Celldifferentiation of various cell lineages is a well documented processand requires no further description herein.

As used herein the term “ex-vivo” refers to cells removed from a livingorganism and are propagated outside the organism (e.g., in a test tube).

Following expansion, for example, the now expanded undifferentiatedhemopoietic stem cells or progenitor cells can be isolated by a varietyof affinity separation/labeling techniques, such as, but not limited to,fluorescence activated cell sorting and affinity separation via anaffinity substrate. Affinity molecules which can be used to implementsuch isolation methods include anti-CD34 antibodies, for example, whichbind CD34+ cells.

According to another aspect of the present invention there is providedanother method of expanding/maintaining undifferentiated hemopoieticstem cells or progenitor cells. The method according to this aspect ofthe-present invention, is effected by implementing the following methodsteps. First, undifferentiated hemopoietic stem cells or progenitorcells are obtained. Second, the indifferentiated hemopoietic stem cellsor progenitor cells are cultured in a medium containing, as a soleingredient or as an additive, a stromal cell conditioned medium, thestromal cell conditioned medium being derived from a stationary phaseplug-flow bioreactor in which a three dimensional stromal cell culture,of either stromal cell line or primary stromal cell culture, have beenestablished on a substrate in the form of a sheet, the substrateincluding a non-woven fibrous matrix forming a physiologicallyacceptable three-dimensional network of fibers, thereby, as is furtherdescribed above and exemplified in the Examples section that follows,expanding/maintaining undifferentiated hemopoietic stem cells orprogenitor cells.

According to yet another aspect of the present invention there isprovided a method of preparing a stromal cell conditioned medium usefulin expanding/maintaining undifferentiated hemopoietic stem cells orprogenitor cells. The method according to this aspect of the presentinvention is effected by implementing the following method steps. First,a stromal cell culture, of either stromal cell line or primary stromalcell culture, is established in a stationary phase plug-flow bioreactoron a substrate in the form of a sheet, the substrate including anon-woven fibrous matrix forming a physiologically acceptablethree-dimensional network of fibers, thereby expanding/maintainingundifferentiated hemopoietic stem cells or progenitor cells. Second,when a desired stromal cell density has been achieved, say, for example,above 5×10⁶ or above 10⁷ cells per cubic-centimeter of the matrix,collecting medium from the stationary phase plug-flow bioreactor, as isfurther described above and exemplified in the Examples section thatfollows, obtaining the stromal cell conditioned medium useful inexpanding/maintaining undifferentiated hemopoietic stem cells orprogenitor cells.

According to still another aspect of the present invention there isprovided a method of transplanting undifferentiated hemopoietic stemcells or progenitor cells into a recipient. The method according to thisaspect of the present invention is effected by implementing thefollowing method steps. First, the undifferentiated hemopoietic stemcells or progenitor cells are expanded/maintained by any of the methodsdescribed above. Second, the undifferentiated hemopoietic stem cells orprogenitor cells resulting from the first step are transplanted in therecipient.

As is shown in FIG. 1, according to yet an additional aspect of thepresent invention there is provided a bioreactor plug comprising acontainer 5, typically in the form of a column, having an outlet and aninlet and containing therein a substrate in the form of a sheet, thesubstrate including a non-woven fibrous matrix forming a physiologicallyacceptable three-dimensional network of fibers, the substrate supportingat least 5×10⁶ stromal cells, preferably, at least 10⁷, of eitherstromal cell line or primary stromal cell culture, per cubic centimeterof the substrate.

According to still an additional aspect of the present invention thereis provided a plug-flow bioreactor comprising the above bioreactor plug.

It will be appreciated in this respect that the substrate maytheoretically support up to 5×10⁷ cells per cubic centimeter thereof.Once sufficient cells have accumulated on the substrate, means such asirradiation can be employed to cease further cell growth, so as tocontrol the exact number of cells supported by the substrate.

The undifferentiated hemopoietic stem cells or progenitor cells whichare used as a source for such cells while implementing the methods ofthe present invention can be purified or isolated from a tissue, suchas, but not limited to, cord blood, cytokine-mobilized peripheral blood(collected by, for example, leukapheresis) and bone-marrow, all of whichare known to include undifferentiated hemopoietic stem cells orprogenitor cells. Methods of such separation are well known in the art,the most frequently used being fluorescence activated cell sorting inwhich cells are first tagged by affinity labeling with a fluorophore andare than collected.

According to a preferred embodiment of the present invention theundifferentiated hemopoietic stem cells or progenitor cells and stromalcells of the stromal cell culture share common HLA antigens. Accordingto another preferred embodiment of the present invention theundifferentiated hemopoietic stem cells or progenitor cells and thestromal cells of the stromal cell culture are from a single individual.Thus, separation of cells is not required in case of transplantationthereof to a recipient.

According to still another preferred embodiment of the present inventionthe undifferentiated hemopoietic stem cells or progenitor cells andstromal cells of the stromal cell culture are from differentindividuals. For example, a future recipient of the undifferentiatedhemopoietic stem cells or progenitor cells and stromal cells be used toprovide the stromal cells, whereas the undifferentiated hemopoietic stemcells or progenitor cells and stromal cells are from a donor selectedaccording to HLA compatibility to donate such cells to the recipient.Thus, again, separation of cells is not required prior totransplantation.

According to another embodiment of the present invention theundifferentiated hemopoietic stem cells or progenitor cells and stromalcells of the stromal cell culture are from the same species. However,according to still another preferred embodiment of the present inventionthe undifferentiated hemopoietic stem cells or progenitor cells andstromal cells of the stromal cell culture are from different species.

According to a presently preferred embodiment of the present inventionthe step of seeding the undifferentiated hemopoietic stem cells orprogenitor cells into the stationary phase plug-flow bioreactor iseffected while flow in the bioreactor is shut off for at least 10 hoursfollowing such seeding, so as to enable the cells to anchor to thestromal cell covered matrix.

According to preferred embodiments of the present invention, culturingthe stromal cells of the present invention is effected under continuousflow of the culture medium. Preferably the flow rate through thebioreactor is between 0.1 and 25 ml/minute, more preferably the flowrate is between 1-10 ml/minute.

The following descriptions provide insight with respect to preferredsubstrates which are used while implementing the present invention.

Thus, according to one embodiment the fibers of the substrate form apore volume as a percentage of total volume of from 40 to 95% and a poresize of from 10 microns to 100 microns. According to another embodiment,the matrix making the substrate is made of fiber selected from the groupconsisting of flat, non-round, and hollow fibers and mixtures thereof,the fibers being of from 0.5 microns to 50 microns in diameter or width.According to still another embodiment, the matrix is composed of ribbonformed fibers having a width of from 2 microns. According to a furtherembodiment, the ratio of width to thickness of the fibers is at least2:1. According to still a further embodiment, the matrix making thesubstrate having a pore volume as a percentage of total volume of from60 to 95%. According to still another embodiment, the matrix has aheight of 50-1000 μm whereas stacks thereof are employed. According toyet another embodiment, the material:of the matrix making the substrateis selected from the group consisting of polyesters, polyalkylenes,polyfluorochloroethylenes, polyvinyl, chloride, polystyrene,polysulfones, cellulose acetate, glass fibers, and inert metal fibers.According to still another embodiment, the matrix is in a shape selectedfrom the group consisting of squares, rings, discs, and cruciforms.According to still another embodiment, the matrix is coated withpoly-D-lysine.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and- Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153, 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

Example 1 Materials and Experimental Methods

Bioreactor: The bioreactor used in accordance with the teachings of thepresent invention was constructed in accordance with the designdescribed in FIG. 1. The glassware was designed and manufactured at theTechnion (Israel) and connected by silicone tubing (Degania, Israel).The carriers were rotated overnight in phosphate buffered saline (PBS;Beit Ha'Emek Industries, Israel) without Ca⁺² and Mg⁺², followed byremoval of the PBS and released debris. Each column was loaded with 10ml packed carrier. The bioreactor was filled with PBS-Ca-Mg, all outletswere sealed and the system was autoclaved (120° C., 30 minutes). The PBSwas removed via container [8] and the bioreactor was circulated in a 37°C. incubator with 300 ml Dulbecco's high-glucose medium (DMEM; GIBCOBRL) containing 10% heat-inactivated fetal calf serum (FCS; Beit Ha'EmekIndustries, Israel) and a Pen-Strep-Nystatin mixture (100 U/ml: 100μg/ml: 1.25 μn/ml; Beit Ha'Emek), for a period of 48 hours. Circulatingmedium was replaced with fresh DMEM containing the above +2 mML-glutamine (Beit Ha'Emek).

Stromal cells: Stromal cell lines were maintained at 37° C. in DMEMsupplemented with 10% FCS, in a fully humidified incubator of 5% CO₂ inair. Cells were grown in tissue culture flasks (Corning) and were splitby trypsinization upon reaching confluence. Primary human marrow stromalcultures were established from aspirated sternal marrow ofhematologically healthy donors undergoing open-heart surgery. Briefly,marrow aspirates were diluted 3-fold in Hank's Balanced Salts Solution(HBSS; GIBCO BRL) and were subject to Ficoll-Hypaque (Robbins ScientificCorp. Sunnyvale, Calif.) density gradient centrifugation. Marrowmononuclear cells (<1.077 gm/cm³) were collected, washed 3 times in HBSSand resuspended in long-term culture (LTC) medium, consisting of DMEMsupplemented with 12.5% FCS, 12.5% horse serum (Beit Ha'Emek), 10⁴ Mβ-mercaptoethanol (Merck) and 10-6 mol/L hydrocortwasone sodiumsuccinate (Sigma). Cells were incubated in 25 ml tissue culture flasks(Corning) for 3 days at 37° C. (5% CO₂) and then at 33° C. (idem) withweekly culture refeeding. Stromal cells from individual donors wereemployed for each bioreactor. For 3D and monolayer studies, primarystromal cell cultures were split by trypsinization (0.25% Trypsin aridEDTA in Puck's Saline A; Beit Ha'Emek) every 10 days, to allowsufficient stromal cell expansion. For LTC-IC and CAFC (see below),stromal cells were irradiated (1500 cGy) using a ¹³⁷Cs source, cultureswere maintained at 33° C. in LTC medium.

Seeding of stromal cells: Confluent cultures of stromal cell lines or5-week primary marrow stromal cells were trypsinized and the cellswashed 3 times in HBSS, resuspended in bioreactor medium (see above),counted and seeded at 10⁶ cells/ml in 10 ml volumes via an injectionpoint ([4], FIG. 1) onto 10 ml carriers in the glass column of thebioreactor. Immediately following seeding, circulation was stopped for16 hours to allow the cells to settle on the carriers. Stromal cellgrowth in the bioreactor was monitored by removal of carriers and cellenumeration by the MTT method (56). When stromal cells were confluent,medium was replaced with LTC medium, for continued studies (preparationof SCM, stem cell seeding).

Preparation of stromal cell conditioned medium (SCM): At equivalent celldensities, monolayer and bioreactor stromal cells were recharged withfresh LTC culture medium. SCM was collected following overnightincubation of the cells. For this purpose, medium flow in the 3Dcultures was stopped for 16 hours and removed directly from the columnprior to re-initiation of circulation. For analysis of the effect ofCD34+ cells on stromal cell production of SRC, circulation was stoppedat various intervals (2-7 days) after seeding of CD34+ into the 3Dsystem and medium collected from the column as described above. SCM wasspun (1000×g, 10 minutes), filtered and stored at −20° C. Stromal cellswere also grown in the bioreactor in serum-free medium, for thecollection of SCM, thereby excluding undefined variables.

Isolation of CD34+ cells: Umbilical cord blood samples taken understerile conditions during delivery were fractionated on Ficoll-Hypaqueand buoyant (<1.077 gr/cm³) mononuclear cells collected. Cells fromindividual CB samples were pooled, incubated with anti-CD34 antibodiesand isolated by midi MACS (Miltenyl Biotech).

Suspension cultures of CD34+ cells: CB CD34+ cells (5×10⁵/well) wereincubated in 24-well dishes (TPP, Switzerland), in 0.5 ml of 0-100% SCM,minus or plus 300 ng/ml each of FLT3 ligand, SCF, or TPO, alone orcombined. Controls contained LTC medium plus or minus cytokines. Cellswere incubated at 37° C. at 5% CO₂ in air. Culture medium was exchangedweekly. Prior to seeding and at various times (1-3 weeks), cells wereharvested, enumerated and assayed for CD34+/38−/CXCR4+ by flowcytometry. Output assays can also include SRC, CAFC and LTC-IC.

Stromal-stem cell cocultures: Isolated, pooled CB CD34+ cells wereseeded at equivalent numbers (about 5×10⁵) onto monolayer or bioreactorcontaining equivalent densities of confluent stromal cells. Uponaddition to the bioreactor, medium flow was stopped for 16 hours toenable contact with stromal cells and was re-initiated at a rate of0.1-1.0 ml per minute. CD34+ cell seeded-stromal cell carriers wereremoved for control studies in the absence of medium exchange.Cocultures were maintained in LTC medium, with or without cytokines. Atvarious times (up to 4 weeks), nonadherent cells were collected frommonolayer supernatants or from circulating culture medium via acontainer ([8], FIG. 1). Adherent cells were collected via sequentialtrypsinization and exposure to EDTA-based dissociation buffer (GIBCOBRL), followed by gentle pipetting of the cells. To avoid the presenceof stromal cells in the resulting suspension, the cells were resuspendedin HBSS+10% FCS and were subjected to a 60 minutes adhesion procedure inplastic tissue culture dishes (Corning), at 37° C. Circulating andcarrier-isolated hemopoietic cells were washed, counted and assayedseparately for CD34+/38−/CXCR4+ by flow cytometry. Output assays canalso include SRC, CAFC and LTC-IC.

Flow Cytometry: Cells were incubated at 4° C. for 30 minutes withsaturating concentrations of monoclonal anti-CD34+PerCP(Beckton-Dickinson), anti-CXCR4-fluorescein isothiocyanate (FITC, R&Dsystems) and—phycoerythrin (PE, Beckton-Dickinson) antibodies. The cellswere washed twice in ice-cold PBS containing 5% heat-inactivated FCS andresuspended for three-color flow cytometry on a FACSscan(Beckton-Dickinson).

LTC-IC and CAFC assays: Freshly isolated CD34+ cells, cells isolatedfrom stromal-stem cell cocultures or from suspension cultures, wereassayed for LTC-IC and CAFC, as previously described (16, 17). Confluentprimary marrow stromal cells were trypsinized, irradiated (1500 cGy) andplated in 0.1 ml in 96-well dishes (Coming) at 1.5×10⁴/well. 24replicate: wells/group were established. Stromal cells were overlaidwith 0.1 ml of LTC medium containing serial dilutions of CD34+ cells(500-5 cells/well), or with serial dilutions of cells harvested fromvarious assays. Cultures were directly incubated at 33° C. for 5 weeks,with weekly half-medium exchange. Plates were spun down at 1000 rpm for10 minutes, culture supernatants removed and remaining cells overlayedwith methylcellulose cultures and cytokines for myeloid progenitor cellassays, as previously described (57). Colonies were enumerated following14 days and LTC-IC frequency determined according to the reciprocal ofthe concentration of test cells that gives 37% negative cultures (16).The CAFC assay was basically performed as above except for the absenceof overlay of methylcellulose and cytokines. The percentage of wellswith at least one phase-dark hemopoietic clone of at least five cells(cobblestone area) beneath the stromal layer was determined at week 6following seeding of the test cell suspensions, in serial dilutions.

Experimental Results

The bioreactor system employed while reducing the present invention topractice is depicted in FIG. 1. It contained four parallel plug flowbioreactor units [5]. Each bioreactor unit contained 1 gram of porrosivecarriers (4 mm in diameter) made of a non woven fabric matrix ofpolyester (58). These carriers enable the propagation of large cellnumbers in a relatively small volume. The structure and packing of thecarrier have a major impact on oxygen and nutrient transfer, as well ason local concentrations and released stromal cell products (e.g., ECMproteins, cytokines, 59). The bioreactor was maintained in an incubatorof 37° C.

The flow in each bioreactor was monitored [6] and regulated by a valve[6 a]. Each bioreactor contains a sampling and injection point [4],allowing the sequential seeding of stromal and hemopoietic cells.Culture medium was supplied at pH 7.0 [13] from a reservoir [1]. Thereservoir was supplied by a filtered [3] gas mixture containingair/CO₂/O₂ [2] at differing proportions in order to maintain 5-40%dissolved oxygen at exit from the column, depending on cell density inthe bioreactor. The O₂ proportion was suited to the level of dissolvedO₂ at the bioreactor exit, as was determined by a monitor [12]. The gasmixture was supplied to the reservoir via silicone tubes. The culturemedium was passed through a separating container [7] which enabledcollection of circulating, nonadherent cells. Circulation of the mediumwas obtained by means of a peristaltic pump [9] operating at a rate of0.1-3 ml/minute. The bioreactor units were equipped with an additionalsampling point [10] and two containers [8, 11] for continuous mediumexchange at a rate of 10-50 ml/day. The use of four parallel bioreactorunits enables periodic dismantling for purposes such as cell removal,scanning electron microscopy, histology, immunohistochemistry, RNAextraction, etc.

In one experiment a bioreactor system containing the murine 14F1.1stromal cell line (24, 60, 61), which was previously shown to supportthe growth of committed human myeloid progenitors (24) has beenestablished. This cell line can also equally support human CB CAFC (FIG.2), LTC-IC (FIG. 3) and CD34+38− cells (FIG. 4), as well as primaryhuman marrow stromal cells. The results presented in these Figures alsoshow that the addition of FLT3 ligand+TPO to these cultures has noeffect on LTC-IC, whereas these cytokines significantly enhanced CAFCand CD34+38− cell output. In contrast, SCF induced a decline in bothLTC-IC and CAFC. When seeded into the bioreactor at 1.5×10⁶ cells/10 mlculture volume, 14F1.1 cells grew and spread on the carriers (FIG. 5).By day 40 following seeding, the carriers contained a 100-fold increasedcell density, i.e., approximately 1.5×10⁶ cells/carrier, 1.5×10⁷cells/ml (Table 1). TABLE 1 Kinetics of 14F1.1 and primary human marrowstroma growth on carriers Time of stromal cells on carrier (days) 14 10human 20 30 40 14F1.1 stroma 14F1.1 14F1.1 14F1.1 Top part 1.5 × 10³ 1.5× 10³   1 × 10⁵ 3.5 × 10⁵ 1.3 × 10⁶ Middle part   1 × 10³ 1.2 × 10³ 1.3× 10⁵ 2.0 × 10⁵ 1.3 × 10⁶ Bottom part   1 × 10³   1 × 10³   7 × 10⁴ 2.0× 10⁵ 1.5 × 10⁶MTT analysis included 5 carriers/determination.Mean of 2 independent experiments.

The cellular density on carriers at various levels of the column was thesame, indicating a homogenous transfer of oxygen and nutrients to thecells. The culture conditions were optimized for these cells: culturemedium (Dulbecco's high-glucose medium+10% fetal calf serum), flow rate(1 ml/min), medium exchange frequency (once a week), initial seedingdensity (as above). No beneficial effect was found for collagen or polyL-lysine carrier coating, on the growth rate and final density of 14F1.1cells. Preliminary findings with primary human marrow stromal cells(Table 1) indicated a similar density of 14F1.1 and primary stromalcells, on days 10 and 14 following seeding, respectively.

In order to assay the functional activity of the stromal cells withinthe bioreactor, the effect of stromal cells conditioned medium (SCM)obtained from the bioreactor column (3D SCM), on the expansion ofCD34+8− cells in suspension cultures seeded with human CB CD34+ cellswas determined. The activity was compared to SCM obtained from monolayercultures (2D SCM) containing the-same concentration of stromal cells. Asshown in FIG. 6, SCM from 14F1.1 cells was found to be equally or morecapable of supporting the maintenance of human CB CD34+38− cells, thanSCM from primary marrow stromal cells. A maximal effect of 14F1.1 SCMwas consistently observed at a lower concentration than that of primarymarrow SCM. Furthermore, 3D SCM was found to be superior to 2D SCM ofboth cell types, in supporting the expansion of human CB CD34+38− cells.The difference in activity between 2D and 3D SCM was more pronouncedwith culture duration (14 versus 21 days). The addition of 14F1.1 3D SCMto suspension cultures of human CB CD34+ cells also resulted in themaintenance of CD34+38−CXCR4+ cells (Table 2), as compared to controlcultures containing medium alone. TABLE 2 Effect of 3D 4F1.1 SCM onyield of CD34+38-/CD34+38-CXCR4+ Cell surface phenotype LTC medium14F1.1 SCM (50%) CD34+38- 370 1296 CD34+38-CXCR4+ 0 38Human CB CD34+ cells (8 × 10⁴/point) were seeded in suspension culturescontaining LTC medium or 50% 3D 14F1.1 SCM.Cultures were harvested 7 days later and cells analyzed by FACS.CD34+38- and CD34+38-CXCR4+ inputs were 2800 and 112, respectively.

Table 3 demonstrates the effect of cytokines in suspension cultures ofCD34+ containing 2D versus 3D SCM. The results clearly demonstrate that3D SCM was superior to 2D SCM in supporting the maintenance of bothCD34+38− and more importantly, the CD34+38−CXCR4+ (SRC) subset. TABLE 3Effect of cytokines on expansion of CD34+38-/CD34+38-CXCR4+ cells in 3D14F1.1 SCM 2D 14F1.1 SCM (50%) 3D 14F1.1 SCM (50%) Cell surface FLT₃FLT₃ phenotype alone ligand + TPO SCF alone ligand + TPO SCF CD34+38-1820 140 0 2720 4080 130 CD34+38- 460 70 0 620 930 0 CXCR4+ CD34+ 37,000178,000 361,000 17,000 25,000 210,000Human CB CD34+ cells (2.6 × 10⁵/point) 50% 2D vs 3D 14F1.1 SCM, in theabsence or presence of FLT3 ligand (300 ng/ml) TPO (300 ng/ml) or SCF(50 ng/ml).Cultures were harvested 7 days later and cells analyzed by FACS.CD34+38- and CD34+38-CXCR4+ inputs were 7900 and 360, respectively.

This may be related to the: stronger effect of 2D SCM on celldifferentiation, as detected by the yield of CD34+ cells. TPO+FLT₃ligand reduced the yield of CD34+38−/CD34+38−CXCR4+ in the presence of2D SCM but enhanced their yield in cultures supplemented with 3D SCM.Again, this can be attributed to the lesser extent of differentiation inthe 3D system, as determined by the CD34+ surface marker. In both 2D and3D SCM cultures, SCF induced a marked increase in stem celldifferentiation and a marked decline in the yield ofCD34+38−/CD34+38−CXCR4+ cells.

In order to assay stromal-stem cell interactions in our bioreactor, themaintenance/expansion of CD34+38− cells on stromal cell (14F1.1)-coatedcarriers was first evaluated. The latter were removed from thebioreactor into silicone-coated 96-well dishes, followed by the additionof CD34+cells. Controls contained carriers alone and carrier-equivalentnumbers of monolayer 14F1.1 cells. As shown in FIG. 7, the survival ofCD34+38− cells was enhanced by the presence of the carrier alone,confirming the beneficial effect of a 3D structure on thesurvival/maintenance of primitive progenitors (36). Stromal-cell coatedcarriers were superior to carriers alone or to monolayer 14F1.1 cells,in promoting the 7-day survival/maintenance of CD34+38− cells. Prolongedculture (day 14) resulted in increased CD34+38− numbers in both 14F1.1monolayer and 14F1.1-coated carrier cultures.

In a subsequent experiment 6×10⁶ pooled CB CD34+ (3×10⁵ CD34+38−) cellswere seeded into a bioreactor containing 4 columns of non-irradiated,14F1.1-coated carriers, in 350 ml circulating culture medium. Mediumflow was stopped for 16 hours and continued thereafter at a normal rate(1 ml/min). Following 4 days of coculture, circulating medium contained10% of the initially seeded CD34+38− cells, determined by FACS analysisof harvested viable cells. Following 18 days of culture, circulatingmedium contained 0.4% CD34+38− cells, while carrier adherent cellscontained 3% of the initially seeded CD34+38− population.

Example 2

Stroma cells and hematopoietic stem cells (HSC) in co-culture spatiallyinteract and form cell-cell contacts. In addition, soluble bioactivefactors secreted by the former could affect growth potential of HSCs.

In order to distinguish between the mechanical support effect and thebiochemical support effect that are both provided by stroma to HSCgrowth, stoma condition medium system was examined. Additionally, theability of stroma-conditioned medium (SCM) produced under variousculture conditions to support growth of HSC was examined.

Materials and Experimental Procedures

Human bone marrow cells—Human bone marrow (BM) samples were collectedfollowing open-heart surgeries according to procedures approved by theInstitutional Review Board of Rambam Medical Center (Haifa, Israel).Samples were collected on-site from the breastbone (sternum),immediately transferred to 50ml sterile tubes containing 1500U Heparin(Kamada, Bet-Kama, Israel) and processed within the next 24 hours.

Cell growth and production of conditioned medium—Human BM primary stromacells or a supporting cell line of fetal liver murine AFT024 cells (ATCC#SCRC) or marrow preadipocytic 14F1.1 (kindly donated by Prof D Zipori,Weizmann Institute of Science, Rehovot Israel 76100) were grown for 8weeks under standard culture conditions (Two Dimensional—2D) or in thePlurix™ Bioreactor (Three Dimensional—3D) under continuous flowconditions (1-5 ml/minute according to the growth phase) to confluence.LTC culture medium supplemented with 12.5% heat-inactivated FCS, 12.5%HS, 10⁻⁴ M glutamine, 10⁻⁴ M mercaptoethanol, 10⁻⁶ M hydrocortisonesodium succinate in the presence of penicillin, streptomycin andnystatin (100U, 100 μg, 1.25 μg per ml, respectively) was replaced everyseven days. Following 8 weeks, medium was collected from the circulationand considered as stroma conditioned medium (SCM). Following collection,SCM was centrifuged (10 minutes at 100×g), pellet was discarded and theresultant SCM was filter sterilized and kept frozen at −70° C. untiluse.

HSC collection and expansion—To obtain HSC for cell expansion, umbilicalcord blood (UCB) was fractionated as follow: samples were diluted 1:4with HBSS (Beit HaEmek, Israel) supplemented with penicillin,streptomycin and Nystatin (100U, 100 μg, 1.25 μg per ml, respectively)and carefully overlaid onto a research-grade Ficoll-Paque solution(d:1.077 g/cm³, Pharmacia Biotech; Uppsala, Sweden; http://www.pnu.com).Blood cells were than separated by 'standard centrifugation (450×g for30 minutes at room temperature). Mononuclear cells (MNC) were recoveredand washed twice in cold HBSS buffer (450×g for 10 minutes). Cellaliquots were spared for enumeration using Turk's solution (1:75 v/vGencyan violet in acetic acid) in Neubauer type hemocytometer (REICHERTJUNG COUNTING CHAMBER, Fisher Scientific Pittsburgh, Pa.). Viabilitycount of MNC was performed using Trypan blue dye exclusion test.

CD34⁺ cells were obtained from MNC after immuno-magnetic separationusing the CD34 midi-MACS selection kit (Miltenyi Biotec; BergischGladbach, Germany; http://www.miltenyibiotec.com). Briefly, 10⁸ MNC wereincubated at 4° C. for 10 minutes in 500 μl MACS buffer (0.5% BSA, 2 mMEDTA in PBS) containing Fc receptor-blocking reagent. Following two washsteps in MACS buffer, cells were incubated in the presence of 100 μlsuper-paramagnetic monoclonal mouse anti-human CD34 nanoparticles. Themixture was left at 4° C. for 30 minutes and washed in MACS buffer for10 minutes at 450×g. Cells were then resuspended in 500 μl MACS bufferand applied to a pre-cooled midi-MACS positive selection-column on amagnet. The column was rinsed with cool MACS-buffer (4×500 μl).Following magnet removal, CD34⁺ cells were eluted with 1 ml of coldMACS-buffer. The enriched CD34⁺ cell fraction was reapplied to anothercolumn for a second selection cycle prior to performing cellenumeration, characterization and viability assays.

FACS analysis—For antigen profiling, cells were washed in FACS buffer(i.e., 5% FCS in PBS). Cells were further incubated for 20 minutes in 4°C. in the presence of 2% human γ-globulins in PBS to block nonspecificFc receptors. Direct immuno-labeling was performed with fluoresceinisothiocyanate (FITC), phycoerythrin (PE) or peridinin chlorophyllprotein (PerCP)-conjugated monoclonal mouse anti-human antibodies (30minutes at 4° C.): CD38-PE (Coulter, Fla., www.beckman.com),CD34-FITC/PerCP and CXCR4-FITC (BD-Pharmingen; San Diego, Calif.;http://www.bdbiosciences.com/pharmingen). Following washing in cold FACSbuffer cells were counted using trypan-blue exclusion method. Cells wereanalyzed and sorted on a FACStar Plus (Becton Dickinson, Calif.)equipped with 5 W argon and 30 MW helium neon lasers. Double and tripleco-labeling experiments were performed with the following antibody mix:double labeling—anti CD34-FITC with anti CD38-PE; triple co-labelingwith:anti CD34-PerCP, anti CD38-PE and anti CXCR4-FITC. Data acquisitionand analysis were performed using LYSIS II software (Becton Dickinson).

The CD34+ sample was initially profiled for expression of CD38 and CXCR4membrane markers using FACS analyses. Briefly, cells were washed in FACSbuffer (5% FCS in PBS). Cells were then further incubated for 20 min in4° C. in human 2% gamma-globulins in PBS to block nonspecific Fcreceptors. Direct immuno-labeling was performed with fluoresceinisothiocyanate (FITC) or phycoerythrin (PE)-conjugated monoclonal mouseanti-human antibodies (30 minutes at 4° C.): CD38-PE (Coulter, Fla.,www.beckman.com) and CXCR4-FITC (BD-Pharmingen; San Diego, Calif.;www.bdbiosciences.com/pharmingen). Following washing in cold FACSbuffer, cells were counted using trypan-blue exclusion method. Cellswere analyzed and sorted on a FACStar Plus (Becton Dickinson, Calif.)equipped with 5 W argon and 30 MW helium neon lasers.

Once identity verified, between 2·10⁴-4·10⁴ CD34+ cells were seeded assuspension cultures in undiluted SCM in presence or absence of acytokine cocktail [300 ng/ml of each of TPO and Flt-3 ligand (FL)].Seven days later, cultures were harvested and hematopoietic cells werecounted and examined using FACS analyses.

Results represent mean±SD of two independent experiments conducted intriplicates. Values are fold expansion of specific hematopoietic cellsduring a 7-day period.

Results

The ability of primary stroma cells conditioned medium (FIGS. 8 a-c) orfetal liver AFT024 conditioned medium (FIGS. 9 a-c) to support expansionof HSC in the presence or absence of added cytokines was tested.

As shown in FIG. 8 a, in the presence or absence of added cytokine,culture medium conditioned by human BM (primary stromal cells) which wasused to sustain expansion of CD34+ cells, exhibited superiority overnon-conditioned medium. However, when expansion of earlier progenitors(CD34+CD38− and CD34+CD38−CXCR4+ cells, FIGS. 8 b and 8 c, respectively)was analyzed under culture conditions that did not use exogenously addedcytokines, it was only the medium conditioned by cultures of BM cellscultivated on 3D matrix that supported undifferentiated cell expansion.

Similar results were obtained with conditioned medium obtained fromstromal cell-line. Culture medium conditioned by AFT024 cells grown on3D matrix demonstrated superiority over media conditioned by either 2Dcultures or non conditioned medium. However, under these cultureconditions, exogenously supplemented cytokines were not inhibitory.

Altogether these results demonstrate that stroma cells conditionedmedium is sufficient to support expansion of hemopoietic stem cells.Furthermore, conditioned medium obtained from 3D culture conditionssupports better cell expansion than conditioned medium obtained from 2Dculture conditions. Finally, culture medium conditioned on 3-D matrix isactive in supporting HSC expansion in culture environment in particularin the absence of added cytokines.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications cited herein are incorporatedby reference in their entirety. Citation or identification of anyreference in this application shall not be construed as an admissionthat such reference is available as prior art to the present invention.

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1. A method of preparing a stromal cell conditioned medium useful inexpanding undifferentiated hemopoietic stem cells to increase the numberof the hemopoietic stem cells, the method comprising: (a) establishing astromal cell culture in a stationary phase plug-flow bioreactor undercontinuous flow on a substrate in the form of a sheet, said substrateincluding a non-woven fibrous matrix forming a physiologicallyacceptable three-dimensional network of fibers, thereby expandingundifferentiated hemopoietic stem cells; and (b) when a desired stromalcell density has been achieved, collecting medium from said stationaryphase plug-flow bioreactor, thereby obtaining the stromal cellconditioned medium useful in expanding the undifferentiated hemopoieticstem cells.
 2. The method of claim 1, wherein stromal cells of saidstromal cell culture are grown to a density of at least 5×10⁶ cells pera cubic centimeter of said substrate.
 3. The method of claim 1, whereinstromal cells of said stromal cell culture are grown to a density of atleast 10⁷ cells per a cubic centimeter of said substrate.
 4. The methodof claim 1, wherein said fibers form a pore volume as a percentage oftotal volume of from 40 to 95% and a pore size of from 10 microns to 100microns.
 5. The method of claim 1, wherein said matrix is made of fiberselected from the group consisting of flat, non-round, and hollow fibersand mixtures thereof, said fibers being of from 0.5 microns to 50microns in diameter or width.
 6. The method of claim 1, wherein saidmatrix is composed of ribbon formed fibers having a width of from 2microns to 20 microns, and wherein the ratio of width to thickness ofthe fibers is at least 2:1.
 7. The method of claim 1, wherein saidmatrix having a pore volume as a percentage of total volume of from 60to 95%.
 8. The method of claim 1, wherein the matrix has a height of50-1000 μm.
 9. The method of claim 1, wherein the material of the matrixis selected from the group consisting of polyesters, polyalkylenes,polyfluorochloroethylenes, polyvinyl chloride, polystyrene,polysulfones, cellulose acetate, glass fibers, and inert metal fibers.10. The method of claim 1, wherein the matrix is in a shape selectedfrom the group consisting of squares, rings, discs, and cruciforms. 11.The method of claim 1, wherein the matrix is in the form of a disc. 12.The method of claim 1, wherein the matrix is coated with poly-D-lysine.13. The method of claim 1, wherein said stromal cells comprise stromalcells of a primary culture.
 14. The method of claim 1, wherein saidstromal cells comprise stromal cells of a cell line.
 15. The method ofclaim 1, wherein said stromal cell conditioned medium is devoid of addedcytokines.
 16. The method of claim 1, wherein a rate of said continuousflow is in a range of 0.1 to 25 ml/minute.
 17. The method of claim 1,wherein a rate of said continuous flow is in a range of 1 to 10ml/minute.